Epi-mode tomographic quantitative phase imaging in thick scattering samples

Abstract

Quantitative phase imaging (QPI) is an important tool in biomedicine that allows for the microscopic investigation of live cells and other thin, transparent samples. Importantly, this technology yields access to the cellular and sub-cellular structure and activity at nanometer scales without labels or dyes. Despite this unparalleled ability, QPI's restriction to relatively thin samples severely hinders its versatility and overall utility in biomedicine. Here we overcome this significant limitation of QPI to enable the same rich level of quantitative detail in thick scattering samples. We achieve this by first illuminating the sample in an epi-mode configuration and using multiple scattering within the sample-a hindrance to conventional transmission imaging used in QPI-as a source of transmissive illumination from within. Second, we quantify phase via deconvolution by modeling the transfer function of the system based on the ensemble average angular distribution of light illuminating the sample at the focal plane. This technique packages the quantitative, real-time sub-cellular imaging capabilities of QPI into a flexible configuration, opening the door for truly non-invasive, label-free, tomographic quantitative phase imaging of unaltered thick, scattering specimens. Images of controlled scattering phantoms, blood in collection bags, cerebral organoids and freshly excised whole mouse brains are presented to validate the approach.

Noninvasive white blood cell quantification in umbilical cord blood collection bags with quantitative oblique back‐illumination microscopy

“…To recover quantitative phase information from the DPC images, we use a fast deconvolution algorithm that accounts for the angular distribution of light at the focal plane of the microscope . Note that optical phase is a function of the wavelength ( λ ), the refractive index change between the sample and the medium (Δ n ), and the thickness of the sample (Δ z ), and are related as described by Equation 2: …”

“…qOBM provides quantitative phase information, but, unlike conventional phase imaging techniques, it can be used in thick scattering environments, including CBUs. 21,22 Traditional phase imaging technologies yield detailed (quantitative) subcellular information without labels or dyes by detecting light transmitted through a thin sample. 23 qOBM extends these capabilities by using an elegant epiillumination scheme 24 and fast reconstruction strategy, 25 packaged into a simple inverted microscope configuration, to achieve the same level of contrast as traditional phase imaging but in thick structures tomographically.…”

“…23 qOBM extends these capabilities by using an elegant epiillumination scheme 24 and fast reconstruction strategy, 25 packaged into a simple inverted microscope configuration, to achieve the same level of contrast as traditional phase imaging but in thick structures tomographically. 21,22 Data from qOBM can also be processed to provide absorptionbased contrast. 22,24 Recently, we have demonstrated the ability of a dualwavelength OBM system to image inside clear blood bags and differentiate between red blood cells (RBCs) and white blood cells (WBCs).…”

“…qOBM provides quantitative phase information, but, unlike conventional phase imaging techniques, it can be used in thick scattering environments, including CBUs . Traditional phase imaging technologies yield detailed (quantitative) subcellular information without labels or dyes by detecting light transmitted through a thin sample .…”

“…Traditional phase imaging technologies yield detailed (quantitative) subcellular information without labels or dyes by detecting light transmitted through a thin sample . qOBM extends these capabilities by using an elegant epi‐illumination scheme and fast reconstruction strategy, packaged into a simple inverted microscope configuration, to achieve the same level of contrast as traditional phase imaging but in thick structures tomographically . Data from qOBM can also be processed to provide absorption‐based contrast …”